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Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: an assay amenable
A M Steff1, M Fortin, C Arguin
1PROCREA BioSciences, Division of Research and Development, Montreal, Quebec, Canada.
Background:
Reliable assessment of cell death is now pivotal to many research programs aiming at generating new anti-tumor compounds or at screening cDNA libraries. Such approaches need to rely on reproducible, easy-to-handle, and rapid microplate-based cytotoxicity assays that are amenable to high-throughput screening (HTS) technologies. We describe a method for the direct measurement of cell death, based on the detection of a decrease in fluorescence observed following death induction in cells expressing enhanced green fluorescent protein (EGFP).
Methods:
Cell death was induced by a variety of apoptotic stimuli in various EGFP-expressing mammalian cell lines, including those routinely used in anti-cancer drug screening. Decrease in fluorescence was assessed either by flow cytometry (and compared with other apoptotic markers) or by a fluorescence microplate reader.
Results:
Cells expressing EGFP exhibited a decrease in fluorescence when treated by various agents, such as chemotherapeutic drugs, UV irradiation, or caspase-independent cell death inducers. Kinetics and sensitivity of this EGFP-based assay were comparable to those of traditional apoptosis markers such as annexin-V binding, propidium iodide incorporation, or reactive oxygen species production. We also show that the decrease in EGFP fluorescence is directly quantifiable in a fluorescence-based microplate assay. Furthermore, analysis of EGFP protein content in cells undergoing cell death demonstrates that the decrease in fluorescence does not arise from degradation of the protein.
Conclusions:
This novel GFP-based microplate assay combines sensitivity and rapidity, is easily amenable to HTS setups, making it an assay of choice for cytotoxicity evaluation.
Insights
A new assay uses enhanced green fluorescent protein (EGFP) to directly measure cell death by detecting a drop in fluorescence. This rapid, sensitive method is ideal for high-throughput screening of anti-cancer compounds.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Accurate cell death assessment is crucial for developing anti-cancer drugs and screening cDNA libraries.
- High-throughput screening (HTS) requires reproducible, rapid, and microplate-based cytotoxicity assays.
- Current methods for cell death detection can be complex or time-consuming.
Purpose of the Study:
- To develop a novel, direct method for measuring cell death.
- To establish a fluorescence-based assay amenable to HTS for cytotoxicity evaluation.
- To utilize enhanced green fluorescent protein (EGFP) for real-time monitoring of cell death.
Main Methods:
- Mammalian cell lines expressing EGFP were treated with various apoptotic stimuli.
- Cell death was assessed by monitoring the decrease in EGFP fluorescence using flow cytometry and a fluorescence microplate reader.
- The EGFP assay was compared with established apoptosis markers like annexin-V binding and propidium iodide incorporation.
Main Results:
- A decrease in EGFP fluorescence was observed in cells undergoing death induced by chemotherapeutic drugs, UV irradiation, and other agents.
- The kinetics and sensitivity of the EGFP assay were comparable to traditional apoptosis markers.
- The decrease in fluorescence was directly quantifiable in a microplate format and did not result from EGFP protein degradation.
Conclusions:
- A novel GFP-based microplate assay offers a sensitive and rapid method for cytotoxicity evaluation.
- The assay is easily adaptable to high-throughput screening (HTS) setups.
- This assay is a valuable tool for anti-cancer drug discovery and research programs focused on cell death mechanisms.